The cellular response to DNA damaging agents involves many genes and pathways. We have taken a systematic approach to identifying all genes that impact on survival/growth response to ionizing radiation. We have now completed the genome wide screening of the total diploid deletion strain collection, for sensitivity to a single dose (80 krads) of ionizing radiation (IR) as previously described (Bennett et al., 2001). The strain collection, which was developed by the Yeast Deletion Project, provides deletions of all nonessential genes or ORFs. OUr recent work completes and extends our previous study in that the ~1200 gene deletions remaining to be screened for radiation sensitivity were also screened for sensitivity to other DNA damaging agents including ultraviolet light (UV), methyl methane sulphonate (MMS), hydroxyurea (HU), bleomycin, camptothecin, and doxorubicin. Among the ~1200 gene deletions, we identified a further 64 deletion strains sensitive to IR. Of these, 58 were not previously associated with sensitivity to any type of DNA damage. Therefore, a total of 165 new genes (58 + 107 previously described) are identified that are required for toleration of IR damage. The study was validated by the identification of 31 genes that were previously well-characterized recombination or checkpoint genes required for radiation resistance, such as RAD52 and RAD9. We estimate that ~4% (196/4746) of the nonessential genes in the yeast genome contribute to IR damage toleration. This estimate is low since essential genes are not included some genes may be part of redundant pathways. Of the 165 new genes, 119 correspond to genes for which a function or genetic role has been suggested based on experimental evidence. Among these genes 90% show cross sensitivity to one or more of the damaging agents described above (Bennett et al., 2001). Based on the cross sensitivities to other DNA damaging agents, we grouped these new gamma-ray resistance genes into 24 functional groupings of which 6 contain previously identified DNA damage or checkpoint repair genes. These functional categories include nuclear pore formation, chromatin structure, vacuolar function, chromosome transmission, transcription, cytoskeleton functions, mitochondrial functions and protein synthesis. Some of the newly identified IR sensitive gene deletions (15) show sensitivities similar to those of known recombinational repair or checkpoint genes. Members of the RAD52 group of DSB repair genes were sensitive to allthe individual DNA damaging agents tested. We have also investigated spontaneous damage-inducible responses in some of the mutant. We found spontaneous expression of the DIN::LacZ reporter plasmid for 7 (ANC1, VID31, CNM67, CTF4, YLR235C, MMS22 and CDC40) of the 21 IR resistance gene deletion strains that showed spontaneous G2 arrest when compared to WT (Bennett et al., 2001). The enhanced transcriptional activation of the DIN promoter as compared to WT indicates the possible presence of persistent damage. All of these genes as well as ASF1, POL32 and VID21 showed enhanced expression of the DIN::LacZ reporter in the presence of continuous low dose exposure to MMS when compared to WT. Six of these gene deletion strains (ANC1, VID31, CNM67, CTF4, YLR235C and MMS22) also showed enhanced activation of the DIN::LacZ reporter following continuous low dose exposure to bleomycin. Surprisingly, four gene deletions (CDC40, ASF1, POL32 and VID31) that showed an enhanced DIN::LacZ response following MMS treatment, did not show DIN::LacZ signaling in response to bleomycin. Since previous epistasis analysis has placed CDC40 as a member of the RAD6 epistasis group, this suggests that postreplication repair (PRR) may be an effector of S phase checkpoint arrest and that CDC40 may play a critical role linking the two processes. Furthermore, it suggests that deletions of ASF1, POL32 and VID31, which all show a signaling response similar to the CDC40 deletion, may participate in PRR as well. All strains that showed enhanced DIN:LacZ responses to either MMS and/or bleomycin also demonstrated reduced survival to these agents. The dun1 checkpoint signaling gene was initially found through its inability to induce RNR genes following DNA damage by agents that are S phase specific (i.e. MMS or HU). Similar to dun1 strains, five strains deleted for LHS1, YJL193W, EST1, HFI1 and DOC1 all showed a decreased DIN::LacZ signaling response following treatment with MMS and/or bleomycin when compared to WT. Three gene deletion strains (EST1, HFI1 and DOC1) are deficient in spontaneous activation of DIN::LacZ and two of these (HFI1 and DOC1) are deficient in damage-induced signaling as well. Surprisingly, of these 5 deletion strains, only the hfi1D showed reduced survival following MMS treatment. These results suggest that these IR resistance genes may, like DUN1, play a transducing role in DNA damage signaling. ASF1 and ANC1 have both been implicated in the S phase checkpoint response. Deletions of either gene is characterized by enhanced DIN::LacZ signaling following replicative (MMS) damage but only the anc1D shows the presence of spontaneous damage signaling suggesting that enhanced MMS-induced DIN::LacZ signaling may be a more diagnostic test of involvement in S phase checkpoint activity. If spontaneous, or low levels of MMS-induced damage persists in these strains due to the lack of proper S phase checkpoint control, then disruption of either repair and/or G2 arrest functions may have a deleterious effect on cell survival in these mutants. We have begun to address these questions by constructing isogenic diploid double deletion strains that combine deletions in the putative S phase checkpoint mutants with either a rad52D or rad9D. This will allow us to examine epistatic relationships in response to IR and S phase specific agents such as MMS and HU. Epistasis analysis is very useful in determining whether two radiation resistance genes reside in the same repair pathway. Genes are epistatic (in the same pathway) if one mutant gene masks the radiation sensitivity of a second mutant gene. Haploid and diploid double deletion strains that contained a rad52, 50 or rad 9 deletion were constructed for a small number of deletion mutations. The ability to obtain viable haploid or diploid double deletions of asf1, mms22 and lip5 with either rad9 or rad52 indicates that when these genes are simultaneously inactivated they are not synthetically lethal. The inability to obtain cnm67 or vid31 with rad9 or rad52 suggests either there are gene dosage effects in meiosis when combining these alleles or alternatively, genetic rearrangements which inhibit meiosis, may be present in the cnm67D and vid31D haploid strains. The diploid asf1, mms22 and lip5 double deletion diploid strains, in combination with either the rad9D or the rad52D alleles, showed a high degree of chromosome loss. Since inactivation of either RAD9 or RAD52 has been associated with elevated levels of chromosome loss, these genes in combination with asf1, mms22 or lip5 appear to further enhance chromosome instability. These diploid double deletions have been used for preliminary epistasis analysis to determine their involvement in either the RAD9 or RAD52 repair pathways. When any of the three genes were deleted in combination with a rad9D and subjected to IR, no enhanced radiosensitivity for cell killing was observed when compared to the rad9D alone suggesting that these genes may be part of the rad9-dependent checkpoint response pathway. Preliminary data further suggests that deletion of ASF1 (but not MMS22 or LIP5) in combination with a rad52D resulted in enhanced radiosensitivity following IR damage when compared to rad52D alone. This suggests that ASF1 may be a member of a DSB repai